mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Exome Sequencing of innate immune system genes in Behçet Disease
Behcet's disease is a chronic disease that causes ulcers in the mouth and/or on genitals as well inflammation in the eye of affected patients. It was first described by the Turkish dermatologist, Hulusi Behcetin 1937. Behcets disease is more common in people of Middle Eastern or Asian descent than in people of European descent (1,3). It affects multiple organ systems and tissues and its presentation varies from patient to patient and so does the age of initial onset (3). In individuals of Middle eastern descent, more males than females are affected while the opposite holds true for individuals of asian descent. Presentation of Disease The cause of of Behcet's disease is unknown but it is suspected to involve an autoimmune response. Symptoms vary in severity and affected area but are usually due to inflammation of blood vessels. These symptoms include (1): *Arthritis *Genital sores *Mouth Sores *Arthritis *Uveitis *Skin problems and on rare occasion, *Meningitis Diagnosing Becet's disease is difficult due to the time of occurence of the symptoms. By documenting symptoms when they occur and how frequently they occur doctor's can diagnose patients. Symptoms often occur as isolated events which appear unrelated unless there is a history of the different symptoms that a particular patient has experienced over time. In addition, symptoms and their presentation can vary from patient to patient. However, key symptoms that are used to diagnose Behcet's include (but are not limited to): #Mouth sores that occur three times over a period of 12 months #Severe inflammation of the eyes #Positive prick test #and a few others #In complicated cases IgD serum levels and HLA phenotypes help distinguish Behcet's disease from other diseases with similar symptomps (7) Exome Sequencing (targeted exome capture) The selective sequencing of exons (protein coding regions of a gene) and their untranslated regions throughout the genome is refered to as exome sequencing . Exome sequencing is useful for the identification of rare disease associated variants in genes that are otherwise missed in GWAS studies due to the low frequency in the population (2,4). Such in depth sequencing of exons and their untranslated regions helps in the identifciation of variations in fuctional genes that are responsible for Mendelian and non- Mendelian diseases alike. Mutations in the coding sequence of a gene that result in changes in the amino acid sequence of the protein product (referred to as non-synonymous substitutions or variation (NSV)) are often the targets of exome sequencing. Because these mutation often result in nonsense mediated decay of the transcripts they can be missed by traditional protein sequencing methods. Exome sequencing also enable the detection of variation ast the site of splicing in the exome that may be involved in the pathology of a disease (5). However, unlike whole genome sequencing which detects variations in both coding and non-coding sequences of genes, exome sequencing can only detect variations in the coding regions of the genes due to the fact that it only targets the 1% of the entire genome that codes for protein. Due to the relatively small size of an exon in comparison to the whole genome, sequencing facilities are able to obtain a more in depth coverage of the nucleotide sequence for that region of the genome by sequencing the same area x times (eg. 10X sequencing). Before sequencing, genes of interest are enriched using PCR probes or molecular inversion probes (figure 2) (5-6). Once the targets have been captured, they are subsequently sequenced and interpreted. Exome sequencing of innate immune genes in Behcet's Disease Genome wide association studies have linked common variations in the human leukocyte gene B51 (HLA-B51) (7), class I major histocompatibility complex genes as well as the genes for IL-10, IL23R-IL-12RB2 with Behcet's disease in turkish and Japanese cohorts (8,9). Additionally, the killer cell lectin-like receptors which are a part of the natural killer gene complex (KLRK1, KLRC1, KLRC2, KLRC3 and KLRC4), the chemokine receptor 1 gene (CCR1) as well as the STAT4 and endoplasmic reticulum aminopeptidase (ERAP1) genes have also been shown to be associated with Behcet's diseases (10). Although the information regarding the adaptive immune sytem's role is being made clear in Behcet's disease, the role of the innate system not completely understood despite observation that patients with active Behcet's lesions have been shown to have have overactive neutrophils present at the sites of the lesions (7,11). To study the role of the innate immune system in Behcet's disease pathology, Kirino et al. performed deep sequencing on 10 geens identified by GWAS studies (L23R, IL10, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, ERAP1) and 11 genes with roles in innate immunity ( IL1B, IL1R1, IL1RN, the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing protein-3 gene (NLRP3) ,the familial Mediterranean fever gene (MEFV), the TNF receptor super family 1A gene , the proline-serine-threonine phosphatase interacting protein gene , the caspase 1 gene , the PYD and CARD domain gene (PYCARD), the nucleotide-binding oligomerization domain 2 gene , and the toll-like receptor 4 gene (TLR4)] to determine whether rare variations in these genes were associated with the disease (2). 'Results': After sequencing the data obtained were anlayzed by CASAVA 1.6 software to detect presumptive low frequency variants that were also rare. To validate these findings Sanger sequencing was performed on the IL-23R gene identified to have rare and low frequency variations with a p value = 3.9 × 10−4 . All 5 variants that were identified by the CASAVA software were found using Sanger sequencing and no others were detected (Figure 3, Table 1). After validation of the non-synonymous variants the NSVs were subjected to three burden tests which revealed that NSVs for IL-23R were significantly associated with Behcet's while those for IL-1R and TLR4 were only nominally associated using the C-alpha test. The same results were obtained for tthe two remaining burden tests (the data- adaptive sum and the step-up tests) as well (Table 1). Two of the NSVs found in the IL-23R gene were found to be protective for Behcet's disease and Crohn's disease. Additionally, two of the NSVs for TLR4 and NOD2 were also protective for Behcet's but were risk variations for Crohn's disease (Table 2). Conclusion Deep resequencing of the innate immune system genes and genes helped identify TLR4, NOD2 and MEFV as Behcet's disease risk associated alleles. The role of these genes in response to bacterial infections suggested that strong inflammatory responses to bacterial infections may play a role in Behcet's disease pathology. This study also identified IL-23R (which had been initially found to be associated with Behcet's through a GWAS study) variants that were associated with protection from Behcet's disease. from these and other results, the authoirs concluded that ineffective clearance of infecting bacteria as well as overactive inflammatory response may play an important role in Behcet's disease. References #http://www.niams.nih.gov/Health_Info/Behcets_Disease/behcets_disease_ff.asp Accessed 11/8/13 #Kirino Y, Zhou Q, Ishigatsubo Y, Mizuki N et al., (2013) Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behçet disease. PNAS 110(20):8134-9 #http://emedicine.medscape.com/article/1122381-overview #Zhou Q, Laxer R, Pelletier M, Ramaswamy M et al., (2013) Exome sequencing in monogenic Behçet-like disease. Pediatric Rheumatology 11 Suppl 1- Meeting Abstract #http://en.wikipedia.org/wiki/Exome_sequencing #Perkel JM (2013) Exome Sequencing: Toward an Interpretable Genome http://www.sciencemag.org/site/products/lst_20131011.xhtml #Sakane T, Takeno M, Suzuki N and Inaba G (1999) Behçet's Disease N Engl J Med 341:12841291 http://www.nejm.org/doi/full/10.1056/NEJM199910213411707 # Remmers EF, Cosan F, Kirino Y, Ombrello MJ et al., (2010) Genome-wide association study identifies variants in the MHC class I, IL10, and ''IL23R-IL12RB2''regions associated with Behçet's disease Nature Genetics:42:698–702 http://www.nature.com/ng/journal/v42/n8/full/ng.625.html # Mizuki N, Meguro A, Ota M, Ohno S et al., (2010) Genome-wide association studies identify IL23R-IL12RB2 and IL10 as Behçet's disease susceptibility loci.Nature Genetics 42:703–706 http://www.nature.com/ng/journal/v42/n8/full/ng.624.html # Kirino Y, Bertsias G, Ishigastubo Y, Mizuki N et al., (2013) Genome-wide association analysis identifies new susceptibility loci for Behçet's disease and epistasis between HLA-B*51 and ERAP1. Nature Genetics 45(2):202-7 http://www.nature.com/ng/journal/v45/n2/full/ng.2520.html # Ehrlich GE (1997) Vasculitis in Behcet's disease. Int Rev Immunol 14(1):81-8 http://informahealthcare.com/doi/abs/10.3109/08830189709116846